Method of treating abnormal concentrations of TNF α

ABSTRACT

Compounds of the structure ##STR1## wherein R is selected from the group consisting of hydrogen, alkyl radicals of 1-6 carbon atoms, the phenyl radical, and the benzyl radical; and wherein R&#39; is selected from the group consisting of the phthalimido radical and the succinimido radical and of the structure ##STR2## wherein X is CH 2  or C═O; R&#34; is H, --CH 2  CH 3 , --C 6  H 5 , --CH 2  C 6  H 5 , --CH 2  CH═CH 2 , or ##STR3## and hydrolysis products of said compounds wherein R&#34; is H and the piperidino ring or both the piperidino and the imido ring are hydrolyzed are useful for the control of abnormal concentrations of TNF α manifested in septic shock, cachexia and HIV infection without substantially effecting the concentration of other cytokines.

This invention was made with Government support under AI 22616-05 AI07012-25 awarded by the Allergy and Infections Institute. The Governmenthas certain rights in the invention.

RELATED APPLICATIONS

This is a continuation of Ser. No. 07/834,588 filed Feb. 12, 1992, nowabandoned, which in turn is a continuation of Ser. No. 07/655,087 filedFeb. 14, 1991, now abandoned.

BACKGROUND OF THE INVENTION

Debilitation, i.e. loss of weight, strength, vascular weakness, andother symptoms are natural sequelae of many diseases which afflicthumans. These may include, for example bacterial infections such astuberculosis; viral infections, particularly retroviral infectionsincluding HIV infections such as AIDS; various forms of arthritisparticularly rheumatoid and degenerative; ulcerative colitis; regionalenteritis; and the like. Human patients with these symptoms may presentwith an acute condition such as septic shock or with a chronic conditionsuch as cachexia.

U.S. Pat. No. 2,830,991 describes a class of therapeutic agents of thegeneral formula I ##STR4## wherein R is selected from the groupconsisting of hydrogen, alkyl radicals containing 1-6 carbon atoms, thephenyl radical, and the benzyl radical; and wherein R' is selected fromthe group consisting of the phthalimido radical and the succinimidoradical.

The subject matter of this patent and of any other patents orpublications identified in this disclosure are incorporated herein byreference.

Preferred compounds within the scope of the above formula I, for use inthis invention are:

3-phthalimido-2,6-dioxo-1-ethyl piperidine

3-phthalimido-2,6-dioxo-1-phenyl piperidine

3-phthalimido-2,6-dioxo-1-benzyl piperidine

3-phthalimido-2,6-dioxo-1-allyl piperidine

3-phthalimido-2,6-dioxo-piperidine

As described in the patent, the compounds are produced by reacting analiphatic dicarboxylic acid, which contains five carbon atoms in astraight chain, the methylene groups of which are substituted by thesubstituents in accordance with the appropriate general formula, withurea or substitution products thereof or with a primary amine or an acidamide in such manner that water is split off and the ring is closed. Ifan amino group is present in the aliphatic chain, this group must notexist in free form in this stage of the process, since otherwise thereis the danger of this amino group participating in an undesirable mannerin the reaction. Instead of using the dicarboxylic acid, it is alsopossible to employ functional derivatives thereof, such as acid halides,acid esters and acid amides.

Compounds of the glutaminic acid series may be used as startingmaterials for the present invention. In this case also, the acidhalides, esters and amides of glutaminic acid may be employed instead ofthe acid itself. It is known that glutaminic acids tends to form5-membered rings with a free amino group. This reaction is undesirablefor the purposes of the present invention. The amino group musttherefore be substituted or protected prior to the ring-closingreaction. The protection of the amino group may be carried out, whenusing products of the glutaminic acid series, by introducing thephthalyl, succinyl or like radical in a manner known per se. Theproportions of the components used for the ring formation must be suchthat at least 1 mol of the compound yielding the imide nitrogen is usedto one mol of the glutaminic acid component.

The first compound listed above is prepared by reacting 27.7 g. ofN-phthalyl glutaminic acid with 66 g. of a 33% solution of ethyl aminein water and slowly heating in an oil bath 160°-180° C., the mixturebeing maintained at this temperature for 15 to 20 minutes. The reactionproduct is recrystallised from alcohol by fractionation. It melts at209° C.

The last compound listed above prepared by reacting 13 g. of phthalylglutaminic acid anhydride and 6 g. of urea in 75 cc. of absolute xylenefor 4 hours at the boiling point of the mixture. Formation of asublimate takes place with evolution of ammonia and carbon dioxide. Thexylene is then distilled off in vacuo and the residue recrystallizedfrom 95% alcohol by fractionation. In addition to some phthalimide andphthalyl glutamine, the required N₂ -phthalyl glutaminic acid imide isobtained, having a melting point of 269°-271° C.

In the patent, the compounds are disclosed as having low toxicity and asuseful for certain spasmolytic and antihistaminic effects. The compound3-phthalimido-2,6-dioxopiperidine is disclosed as being particularlyuseful as a sedative. This compound was marketed as a sedative under thegeneric name thalidomide. It was subsequently discovered to beteratogenic and was withdrawn from the market.

Despite its teratogenicity, thalidomide has long been employed for thetreatment of erythema nodosum leprosum (ENL) an accute inflammatorystate occurring in lepromatous leprosy. See, for example Mellin, G. W.,and M. Katzenstein. N. Engl. J. Med. 267:1184 (1962). More recently, ithas been shown to be useful in the treatment of graft-versus-hostdisease by Vogelsany, G. B., S. Taylor, G. Gordon and A. D. Hess.Transplant Proc. 23:904 (1986); for treatment of reheumatoid arthritisby O. Gutierrez-Rodriguez, P. Starusta-Bacai and O. Gutierrez-Montes.The Journal of Rheumatology 16:2 158 (1989); and for treatment ofaphthous ulceration in patients positive for HIV antibody. Brit. Med. J.298:432 (1989).

The tumor necrosis factor (TNF-.sub.α) is one of several cytokinesreleased mainly by mononulear phagocytes together with several othercytokines in response to stimuli to the immune system. It is requiredfor a cell mediated immune response to overcome infections. As its namesuggests, it is associated with the destruction of tumor cells. It isnot present in measureable amounts in normal sera, but appears, oftenvery rapidly, in response to immunostimulators such as bacterial andviral infections, particularly HIV infections. In the case of chronicinfection it may be found in the sera at relatively high or low levelsfor extended periods of time. It may also appear suddenly in highconcentrations in response to release of a toxin by an invadingbacteria. It is markedly elevated in ENL.

TNF-.sub.α has been recognized as manifesting a dose dependent toxicity.If present at low levels for too long a period it results in cachexia.At high levels even for a short time it results in septic shock.

Cachexia is a general weight loss and wasting occurring in the course ofa chronic disease. More specifically, it is a weight loss not accountedfor by decreased caloric intake. It is associated with cancer, theopportunistic infections of AIDS, inflammatory diseases, parasiticdiseases, tuberculosis, high dose IL-2 therapy and the like. It is achronic condition related to chronic diseases.

Septic shock is an acute condition usually, but not always attributed toinfection or to toxic substances in the tissue. It is characterized byhypotension due to loss of vascular tone. It may result in patientcollapse, or even death if not treated promptly and efficiently.

The retroviruses are a broad group of RNA viruses which, during theirreplication, employ the reverse transcription enzyme (RT) to convert aRNA message to DNA. The retroviridae family of viruses includeslentiviruses (visna, maedi, progressive pneumonia virus -"slowviruses"), spumaviruses (foamy viruses) and oncornaviruses (types A, B,C, D, RNA tumor viruses). The retroviruses have been shown to infectmurine, avian, feline, primate, and human species.

The human immunodeficiency virus (HIV-1) or human T-Cell lymphotropicviruse (HTLV-III) which causes Acquired Immune Deficiency Syndrome(AIDS), AIDS related complex (ARC) and other AIDS related diseases is aretrovirus. TNF-.sub.α functions in an autocrine manner in the inductionof HIV-1 expression (G. Poli et al, PNAs Vol 87 p 782, 1990).

It is apparent, therefore, that it is necessary to control theconcentration of TNF-.sub.α in the sera to avoid the debilitatingeffects of abnormal concentrations of this cytokine including, forexample, cachexia and septic shock.

Other cytokines which are necessary for a proper immune response arealso produced by mononuclear phagocytes. These include, for example,various interleukins such as IL-1, IL-6, IL-8 and the granulocytemacrophage colony stimulating factor, GM-CSF. Still other cytokines areproduced by the T-cells. It is desirable to control the concentration ofTNF without appreciably affecting the concentration and activity ofother cytokines.

Heretofore, antiinflammatory and immunosuppresive steroids such asprednisolone and dexamethasone have been employed to treat thedebilitating effects of TNF-.sub.α. Unfortunately, these therapeuticagents also block the production of other cytokines so that the patientsbecome susceptible to life threatening infections.

BRIEF SUMMARY OF THE INVENTION

It has now been discovered that the debilitating effects of toxicconcentrations of TNF-.sub.α, whether acute or chronic, can becontrolled in humans by treating a human patient in need of suchtreatment with an anti-debilitating amount of a compound within thescope of the above description. Typically the treatment may be eitheroral or parenteral, for example intravenously or subcutaneously.

It has further been discovered that certain compounds within the scopeof the above formula as well as other closely related compounds areespecially useful for the practice of this invention. These compoundsare presently preferred for the therapeutic purposes of the inventions.These preferred compounds include those represented by formula II##STR5## wherein X is CH₂ or C═O; R is H, --CH₂ CH₃, --C₆ H₅, --CH₂ C₆H₅, --CH₂ CH═CH₂, or ##STR6## and hydrolysis products of said compoundswherein R is H and the peperidino ring or both the peperidino and theimido ring are hydrolyzed.

Especially preferred compounds within the ambit of the above definitionare represented by the formulas: ##STR7##

Most of the non-hydrolyzed compounds whose formulas are given above canbe prepared by the processes described in the aforesaid U.S. Pat. No.2,830,991. The preparation of the phthalimidine compounds is describedin U.S. Pat. No. 3,705,162. U.S. Pat. No. 3,563,986 describes thepreparation of the morpholino substituted compounds. The hydrolyticcompounds are prepared by standard hydrolysis procedures several ofwhich will be known to the skilled artisan.

The compounds used in the invention can exist as racemic mixtures. Theracemic mixtures and separate isomers are included within the scope ofthe invention.

The compounds may be administered alone, but will normally be employedin a composition containing a pharmaceutically acceptable carrier. Itmay be advantageous, as will be discussed more fully below to administerthe selected compound or compounds together with an effective amount ofa therapeutic agent appropriate for treating the cause of the abnormalconcentration of TNF-.sub.α, for example with an antibacterial agent ifthe condition under treatment is shock caused by the sudden release oflarge amounts of a toxin because of bacterial infection.

THE DRAWINGS

FIGS. 1a, 1b, 1c; 2, 3, show the effects of thalidomide on TNF-.sub.αproduction in the presence of various reagents.

FIGS. 4 through 7 show the results of studies conducted to establish theutility of the compounds of this invention to inhibit HIV-1 RT activity.

The drawings and the balance of this disclosure will be betterunderstood by recognizing the meanings of certain abbreviations. CWP-MLmeans cell wall protein of Mycobacterium leprae. ENL means erythemanodosum leprosum. GM-CSF means granulocyte macrophage colony-stimulatingfactor. PPD means purified protein derivative of tuberculin. PBMC meansperipheral blood mononuclear cells.

The studies described hereinafter will be recognized by those skilled inthe art as establishing that the compounds of this invention selectivelyinhibit the production of human TNF-.sub.α without substantiallyaffecting the production of other proteins or of total serum protein.Therefore, although the compounds of the invention will not curediseases, they will significantly improve the quality of life of thepatients. An important consequence of the study is the finding thatTNF-.sub.α secretion is not totally inhibited. This is important since,as indicated above, TNF-.sub.α appears to be an essential mediator inthe immune response.

There follows a complete description of one procedure for establishingthe ability of the compounds of this invention to inhibit the productionof TNF-.sub.α without inhibiting the production of other cytokines.

Monocyte Isolation.

PBMC obtained by Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway,N.J.) density centrifugation were rosetted with neuraminidase-treated(Vibrio cholerae neuraminidase; Calbiochem-Behring Corp. La Jolla,Calif.) sheep erythrocytes (Scott Laboratories, Friskville, R.I.) (SRBCrosetting), and the nonrosetted cells were counted (E⁻ populationmonocytes enriched). 10⁶ cells were cultured at 37° C. in 24-well plates(Corning Glass Works, Corning, N.Y.) in 1 ml of RPMI 1640 (GibcoLaboratories, Grand Island, N.Y.) supplemented with 10% AB⁺ serum, 100U/ml penicillin, 100 μg/ml streptomycin, and 2 mM 1-glutamine. AdherentE⁻ cells were used for the studies.

Cytokine Agonist

LPS of Salmonella minnesota R595 (List Biological Laboratories,Campbell, Calif.) was diluted in PBS, pH 7.4, and used at 1 g/ml;Purified protein derivative of tuberculin (PPD) was purchased fromStatens Seruminstitut, Copenhagen, Denmark; CWP-ML was prepared usingknown and published methods. The concentrations of the stimulatingagents were those known to induce optimal TNF-.sub.α protein productionby cultured monocytes. The endotoxin content of solutions andmycobacterial preparations was estimated by the Limulus amebocyte lysateassay (LAL; Whittaker M. A. Bioproducts, Walkersville, Md.). Allsolutions used contained less than 10 pg/ml of endotoxin.

Cytokine Induction

Adherent E⁻ cells were stimulated with 1 μg/ml of LPS, 10 μg/ml of PPD,or 10 μg/ml of CWP-ML for up to 18-20 h. At various times, supernatantswere harvested, centrifuged to remove cells and debris, and kept frozenuntil use (-20° C.).

TNF-.sub.α Assay

TNF-.sub.α concentration in the supernatants was determined with aTNF-.sub.α specific ELISA, specific for the biologically activemolecule. Assays were performed in 96-well plates (Nunc Immunoplates,Roskilde, Denmark) coated with the affinity-purified rabbitanti-TNF-.sub.α antibody (0.5 μg/ml; 12-16 h; 4° C.) and blocked for 2 hat room temperature with PBS/0.05% Tween 20 (Sigma Chemical Co., St.Louis, Mo.) containing 5 mg/ml BSA. After washing, 100 μl of TNF-.sub.αstandards, samples, and controls were applied to the wells, and theplates were incubated for 12-24 h at 4° C. After the incubation, plateswere washed and a second antibody, horseradish peroxidase(HRP)-conjugated mouse monoclonal anti-TNF-.sub.α, diluted 1:2,000 inPBS/BSA/Tween, was applied to the wells and incubated for 2 h at roomtemperature. The color reaction was developed with the OPD substrate(0.4 mg/ml o-phenylenediamine [Sigma Chemical Co.] in 24 mM citric acid,51 mM sodium phosphate, pH 5.0 [phosphate-citrate buffer: Sigma ChemicalCo.] containing 0.012% hydrogen peroxide [H₂ O₂ ; Fisher Scientific Co.,Pittsburgh, Pa.]) and absorbance read at 492 nm in an automated ELISAreader (Dynatech Laboratories, Inc., Alexandria, Va.).

IL-1 Assays

IL-1 levels were determined using a commercial ELISA kit (CistronBiotechnology, Pine Brook, N.J.) according to the manufacturer'sspecifications. IL-1 levels are expressed as pico-grams per milliliterof protein.

IL-6 Assay

IL-6 levels were determined using a biological assay as described byFinkelman et al. Proc. Natl. Acad. Sci. USA. 83:9675 (1986).Proliferation of 7TD1 hybridoma cell line specifically sensitive to IL-6was measured by colorimetric determination of hexosaminidase levels,Laudegren et al J. Immunol. Methods. 67:379 (1984), and values for IL-6in the samples were obtained by interpolation from a standard curve. 1U/ml of IL-6 corresponds to the concentration that yields half-maximalgrowth.

Granulocyte/Macrophage CSF GM-CSF Assay

GM-CSF levels were determined using a commercial ELISA kit (Genzyme,Boston, Mass.) according to the manufacturer's specifications, and wereexpressed as picograms per milliliter of protein.

Thalidomide Inhibition

The thalidomide used in this study was the purified drug (racemicmixture: D[+] and L [-] forms) (lot No. JB-I-114; Andrulis ResearchCorporation, Beltsville, Md.). The compound was shown to be at least 99%pure, as analyzed by Fourier Transform Infrared Spectrum. It was thendiluted in DMSO (Sigma Chemical Co.); further dilutions were done insterile PBS.

Percentage inhibition of TNF-.sub.α secretion was calculated as:100×[1-(TNF-.sub.α experimental/TNF-.sub.α control)]; where TNF-.sub.αexperimental represents TNF-secretion by stimulated monocytes that werecultured in the presence of thalidomide, and TNF-.sub.α controlrepresents TNF-.sub.α secretion by stimulated monocytes that werecultured in the absence of the drug. Monocytes cultured in mediumcontaining equivalent amounts of DMSO in the presence or absence of thestimulating agent were used as controls for thalidomide-treated cells.Neither thalidomide nor DMSO had any effect on cell viability orfunction at the concentrations used.

Protein Synthesis

Human monocytes were cultured in Teflon beakers in methionine-free RPMIwith 10% AB⁺ serum at 37° C. for 1 h, when 200 μCi/ml³⁵ S-methionine(1,153 μCi/mmol; ICN Biomedicals Inc., Calif.) was added to the culturesfor the next 3 h with or without the stimulating and the suppressiveagent. At the end of the labeling period, ³⁵ S-labeled cells were washedtwice in ice-cold PBS and lysed directly in 500 μl lysis solution (10 mMTris-HCl buffer, pH 7.4 150 NaCl, 1 mM EDTA, and 1% SDS). Resolving 8%SDS-PAGE was performed overnight. The gel was washed, dried, andanalyzed by autoradiography at -70° C. using XAR-5 radiographic film(Kodak, Rochester, N.Y.) with an intensifying screen.

RESULTS OF THIS STUDY

Monocytes were enriched from PBMC of normal donors and stimulated invitro for 18-20 h with bacterial LPS and mycobacterial products, knownagonist of monocyte TNF-.sub.α synthesis and secretion. Thalidomidesuppressed LPS-stimulated TNF-.sub.α production (FIG. 1A) with a 50%inhibitory concentration (IC₅₀) of 1-4 μg/ml, and 90% inhibitionobserved at 10 μg/ml (18-20-h assay). Similar results were obtained whenPPD and CWP-ML were used as stimulants (FIG. 1, B and C, respectively).

FIG. 1 shows the effect of thalidomide on (A) bacterial endotoxin (LPS,1 μg/ml), (B) PPD, (10 μg/ml), and (C) CWP-ML (10 μg/ml)- inducedTNF-.sub.α production. Monocytes were stimultaneously incubated with 2ng/ml to 10 μg/ml of thalidomide in the culture medium. Control cellswere cultrued in medium alone. A dose-dependent inhibition of TNF-.sub.αsecretion by thalidomide is apparent. No detectable production ofTNF-.sub.α protein was observed in supernatants of unstimulatedmonocytes. Data represent mean ± SD of 15(A), two (B), and one (C)different experiments, respectively.

The inhibition of TNF-.sub.α secretion by thalidomide was dependent uponthe state of monocyte stimulation as shown in Table 1. Preincubation ofunstimulated monocytes with thalidomide, followed by removal of the drugbefore LPS stimulation, did not lead to suppression. By comparison, whenLPS and thalidomide were added stimultaneously to the cultures,irreversible suppression occurred, even when the drug was removed aftera few hours (Table 1). Therefore, the thalidomide sensitive reaction(s)occurs only after the LPS induction of TNF-.sub.α production.

                  TABLE 1                                                         ______________________________________                                        h                   h                                                         ______________________________________                                        A   0-4       0     0     4-20 0     +   100                                  B   0-4       +     0     4-20 0     +   90 ± 4.6                          C   None      0     0     0-4  +     +   48 ± 15                           D   0-4       +     +     4-20 0     +   56 ± 0.5                          E   None      0     0     0-20 +     +   52 ± 9.3                          ______________________________________                                    

Human monocytes cultured in 24-well plates were preincubated with theinhibitory drug with or without the stimulating agent. After 4 h, thecultures were washed, medium was replaced, and LPS was added again forthe next 16 h. Culture supernatants were recovered at the differentperiods and TNF-.sub.α levels determined as described. LPS-inductedrelease of TNF-.sub.α by monocytes cultured for 20 h in the absence ofthalidomide (A). No inhibitory action of thalidomide was detected whenthe drug was washed away before the addition of the stimulating agent(B). Thalidomide-induced inhibition of TNF- production in the present ofLPS after 4 h of stimulation (C), which persisted even after the drugwas washed away (D). Control experiment in which thalidomide was kept inthe cultures with the stimulating agent during the whole assay (E). Datarepresent mean ± SD of two different experiments.

The inhibition of LPS-stimulated TNF-.sub.α secretion by thalidomideoccurs in a setting in which many other proteins are being synthesizedby both constitutive and induced mechanisms. Thus, a simple explanationfor the effect of the drug on TNF-.sub.α production could be asuppression of overall protein synthesis.

FIG. 2 illustrates the effect of thalidomide on the pattern and quantityof proteins synthesized after a 3-h pulse of ³⁵ S-methionine. The totalincorporation of isotope into TCA-precipitable proteins as well as theintensity of most of the individual bands on SDS-PAGE of LPS-triggeredmonocytes remained unchanged after thalidomide treatment.

In FIG. 2 can be seen the effect of thalidomide on protein synthesis byhuman peripheral blood monocytes. Electrophoretic analysis of lysatesfrom monocytes incubated with ³⁵ S-methionine was performed. Cells werestimulated in vitro with and without LPS in the presence or absence ofthalidomide at 1 and 4 μg/ml. TCA-precipitable radioactivity (10% TCAprecipitation) was measured by liquid scintillation counting. The amountof radioactivity in the pellets expressed as cpm×10⁻³ and represents themean of three precipitates with a SD of 10%. Neither total radioactivitynor the pattern of most of the protein bands in the gel was affected bythalidomide (lane 1) unstimulated cells, 3.3×10⁻² cpm in TCAprecipitates; (lane 2) cells stimulated with 1 μg/ml LPS, 4.2×10⁻² cpmin TCA precipitate; (lane 3) cells stimulated with LPS in the presenceof 1 μg/ml thalidomide, 4.2×10⁻² cmp in TCA precipitate; (lane 4) cellsstimulated with LPS in the presence of μg/ml thalidomide, 4.1×10⁻² cpmin TCA precipitate; (lanes 5 and 6) cells incubated only withthalidomide at 1 or 4 μg/ml, respectively, 3.2×10⁻² and 2.8×10⁻² cpm inTCA precipitates, respectively.

Several cytokines are produced by monocytes in response to LPS inaddition to TNF-.sub.α, including IL-1 and IL-6. FIG. 3 shows thatthalidomide exerts a selective effect by suppressing only TNF-.sub.αsecretion LPS-stimulated monocytes. Whereas 4 μg/ml thalidomidesuppressed TNF-.sub.α production (41.9% inhibition) (FIG. 3A), neitherIL-1 (FIG. 3B), IL-6 (FIG. 3 C), nor GM-CSF production (FIG. 3 D) wasinfluenced by the drug. Similar but more extensive selective suppressionwas observed with much higher (up to 20 μg/ml) concentrations ofthalidomide. It was also observed that the D (+) enantiomer appeared tobe more active than the L(-) enantiomer.

FIG. 3 shows the levels of different cytokines tested in culturesupernatants of human monocytes stimulated with LPS for 6 h (A-C) or 20h (D) in the presence or absence of 4 or 10 μg/ml of thalidomide. Datarepresent mean ± SD of six different experiments for TNF-.sub.α and IL-1determinations and three experiments for IL-6 and GM-CSF measurements.About 41.9±14.6% and 52.8±14.7% inhibition of TNF-.sub.α secretion wasfound in the presence of 4 and 10 μg/ml of thalidomide, respectively."Cont" illustrates unstimulated cells cultured in medium. No effect onIL-1, IL-6, or GM-CSF secretion was detected in these cultures.

The following study establishes the utility of compounds of theinvention for reducing TNF-.sub.α concentration in HIV infections.TNF-.sub.α is known to induce HIV replication. Similarly, it is knownthat peripheral blood monocytes from HIV infected patients secretehigher amounts of TNF-.sub.α than do monocytes from uninfectedindividuals. TNF-.sub.α is a cytokine capable of inducing viralexpression in cells chronically infected with HIV. The art, therefore,has long been concerned with discovering products capable of inhibitingTNF-.sub.α production in HIV infected patients. The compounds of thisinvention are capable of so doing. This fact was established in studiesusing the known and commercially available chronically infected celllines U1 and ACH-2, a promonocytic cell line and a T-lymphocytic cellline. The procedure employed is described by Poli et al. (1990) Proc.Nat'l. Acad. sci. U.S.A. Vol. 87, pp 782-785.

Briefly, the expression of HIV was upregulated by the addition of 10⁻⁷ mof phorbol 12-myristate 13-acetate (PMA) or 1 μg/ml of TNF.sub.α toACH-2 and U1 cells. The cells were suspended at 4×10⁵ per ml in RMP11640 medium (M.A. Bioproducts) supplemented with 10% (vol/vol) fetalcalf serum in the presence of the selected amount of stimulator at 37°C. in 5% CO₂ /95% air for 48 hours, the supernatants collected andtested for the presence of Mg⁺⁺ dependent reverse transcriptase activityusing the procedure of Willy et al (1988) J. Virol. 62, 139-147.

For the test, 10 μl of supernatants were added to 50 μl of a mixturecontaining 5 μg per ml of poly(rA) p(dT) 12-18, (Pharmacia), 5 mM MgCl₂and 10 μci/μl of ³² P-labeled deoxythymidine 5'- triphosphate(dTTP-Amersham), and the mixture was incubated for 11/2 hours at 37° C.Eight microliters of the mixture were spotted onto DE81 paper (Whatman),air-dried and washed 5 times in 2×standard saline citrate buffer, andtwo additional times with 95% ethanol. The paper was dried, cut andradioactivity assayed. The results are shown in the figures.

FIG. 4 shows the results of tests in which 5, 10 and 50 μg/ml ofthalidomide (THAL) and the known TNF.sub.α inhibitor pentoxyfylline(PTN) were used to inhibit reverse transcriptase production with thecell line U1. For the comparison, reverse transcriptase activity in theabsence of the inhibitor was taken as 100%. It will be seen that at aconcentration of 50 μg/ml, thalidomide was as effective as PTN.

FIG. 5 shows the results of a similar test with a U1 cell linestimulated with PMA comparing thalidomide and PTN with other compoundsof the invention including the D isomer of thalidomide. The othercompounds of the invention are identified in this and the followingfigures by the letters used under their formulas hereinabove.

FIG. 6 shows a similar study in which the same compounds were testedwith ACH-2 stimulated with TNF-.sub.α.

FIG. 7 records the results of a test using the ACH-2 cell linestimulated with PMA.

The compounds of the invention or their pharmaceutically acceptablesalts may be administered perorally in a pharmaceutical carrier instandard form such as tablets, pills, lozenges, dragees and similarshaped and/or compressed preparations. It is also possible to produceemulsions or suspensions of the compounds in water or aqueous media suchas unsweetened fruit juices and by means of suitable emulsifying ordispersing agents. They may also be employed in the form of powdersfilled into gelatin capsules or the like.

Such powders and mixtures for use in the preparation of tablets andother shaped and/or compressed preparations may be diluted by mixing andmilling with a solid pulverulent extending agent to the desired degreeor firmness or by impregnating the already milled, finely powdered,solid carrier with a suspension of the compounds in water or with asolution thereof in an organic solvent and then removing the water orsolvent.

When preparing tablets, pills, dragees, and the like shaped and/orcompressed preparations, the commonly used diluting, binding, anddisintegrating agents, lubricants, and other tableting adjuvants areemployed, provided they are compatible with agent to be administered.Such diluting agents and other excipients are, for instance, sugar,lactose, levulose, starch, bolus alba; as disintegrating and bindingagents, gelatin, gum arabic, yeast extract, agar, tragacanth, methylcellulose, pectin: and as lubricants stearic acid, talc, magnesiumstearate, and others.

They may be administered in the form of suppositories, typicallyutilizing such commonly used suppository vehicles, as cocoa butter.

The compounds may also be administered parenterally employing aqueoussolutions or suspensions of watersoluble compounds or suspensions. Thecompositions may be made isotonic e.g. with salt or other solute and maycontain a buffer, for example a phosphate buffer.

As indicated above, the compound employed in the invention may be theonly active ingredient administered or it may be coadministered withanother therapeutic agent in an amount which is effective to treat thecondition associated with the debilitating effect. For example, if thecause of the condition is a toxin released by an infectious bacteria, anantibiotic such as tetracycline, penicillin, streptomycin and the likemay be coadministered. If there is hypotension associated with lack ofvascular tone, a vasopressive agent such as epinephrine or dopamine maybe coadministered. If the patient is under treatment with achemotherapeutic agent such as adriamycin, the compound of the inventionand the chemotherapeutic agent may be coadministered.

The term "coadministered" does not mean that the compound of theinvention and the additional therapeutic agent are administered in thesame dosage unit, although they may be so administered. It means thatthey are administered within the same time span.

An "effective amount" of the compound or additional therapeutic agentwill vary with the condition being treated, the age, weight and generalphysical condition of the patient under treatment and other factorsreadily evaluated by the physician in attendance.

What is claimed is:
 1. The method of treating the toxic symptoms of highconcentrations of TNF.sub.α manifested in septic shock, cachexia, andHIV infection by inhibiting the production of TNF.sub.α which comprisesadministering to a human susceptible to or exhibiting such symptoms aneffective amount of a compound of the formula: ##STR8## in which R ishydrogen, alkyl of 1 to 6 carbon atoms, phenyl, or benzyl, andR' is##STR9##
 2. The method of claim 1 wherein R' is ##STR10##
 3. The methodof claim 2 wherein said compound is 3-phthalimido-2,6-dioxopiperidine.4. The method of claim 2 wherein said effective amount is sufficient toproduce a blood level of said compound of at least 0.1 μg/mL.
 5. Themethod of treating the debilitating effects of septic shock caused byhigh concentrations of TNF.sub.α by inhibiting production of TNF.sub.αwhich comprises administering to a human susceptible to or exhibitingsuch effects an effective amount of a compound of the formula: ##STR11##in which R is hydrogen, alkyl of 1 to 6 carbon atoms, phenyl, or benzyl,andR' is ##STR12##
 6. The method of claim 5 wherein R' is ##STR13## 7.The method of claim 6 wherein said compound is3-phthalimido-2,6-dioxopiperidine.
 8. The method of claim 6 wherein saideffective amount is sufficient to produce a blood level of said compoundof at least 0.1 μg/mL.
 9. The method of treating the debilitatingeffects of cachexia caused by high concentrations of TNF.sub.α byinhibiting production of TNF.sub.α which comprises administering to ahuman susceptible to or exhibiting such effects an amount of a compoundof the formula: ##STR14## in which R is hydrogen, alkyl of 1 to 6 carbonatoms, phenyl, or benzyl, andR' is ##STR15##
 10. The method of claim 9wherein R' is ##STR16##
 11. The method of claim 10 wherein said compoundis 3-phthalimido-2,6-dioxopiperidine.
 12. The method of claim 10 whereinsaid effective amount is sufficient to produce a blood level of saidcompound of at least 0.1 μg/mL.
 13. The method of treating thedebilitating effects of an HIV infection caused by high concentrationsof TNF.sub.α by inhibiting production of TNF.sub.α which comprisesadministering to a human susceptible to or exhibiting such effects anamount of a compound of the formula: ##STR17## in which R is hydrogen,alkyl of 1 to 6 carbon atoms, phenyl, or benzyl, andR' is ##STR18## 14.The method of claim 13 wherein R' is ##STR19##
 15. The method of claim14 wherein said compound is 3-phthalimido-2,6-dioxopiperidine.
 16. Themethod of claim 14 wherein said effective amount is sufficient toproduce a blood level of said compound of at least 0.1 μg/mL.
 17. Themethod of treating the toxic symptoms of high concentrations ofTNF.sub.α manifested in septic shock, cachexia, and HIV infection byinhibiting the production of TNF.sub.α which comprises administering toa human susceptible to or exhibiting such symptoms an effective amountof a compound of the formula: ##STR20## in which R is allyl ormorpholinomethyl, and R' is ##STR21##
 18. The method of claim 17 whereinsaid effective amount is sufficient to produce a blood level of at least0.1 μg/mL.